Figure 8

Exposure to phosphoramide mustard, cisplatin or doxorubicin leads to increased expression of phosphorylated H2AX. Representative photomicrographs of cultured testis fragments showing immunohistochemical localisation of γH2AX (green) of cultured testis tissue collected 16 h after the start of drug exposure, counterstained with DAPI (blue). (A) Analysis of γH2AX expression using ImageJ/Fiji software. (Ai) Photomicrograph showing cells with high intensity foci (arrow) and with low intensity foci (arrowhead) of γH2AX. (Aii) Black and white image of selected areas containing only cells with high intensity foci (arrow), after setting a high intensity threshold to exclude cells with low intensity foci (arrowhead) alongside a minimum particle size to exclude signals too small to be cells. (Aiii) Image showing the selected areas (black areas in Aii) from which pixel intensity is measured to obtain value of integrated density. (B–E) Representative images showing γH2AX expression from each group; (B) Control, (C) Mid PM, (D) Low CIS, (E) Low DOX. (F) Intensity of γH2AX expression normalised for tubule area; n = 6 in all groups. Scale bars represent 50 µm. Data are mean + SEM; p < 0.05 (*), p < 0.001 (***) for treatment versus Control.